Blood, 15 April 2003, Vol. 101, No. 8, pp. 3316-3318
BRIEF REPORT: RED CELLS
Duodenal nonheme iron content correlates with iron stores in
mice, but the relationship is altered by Hfe gene
knock-out
Robert J. Simpson,
Edward S. Debnam,
Abas H. Laftah,
Nita Solanky,
Nick Beaumont,
Seiamak Bahram,
Klaus Schümann, and
S. Kaila S. Srai
From the Department of Life Sciences, King's College
London, England; Departments of Physiology and Molecular
Biology, Royal Free and University College School of Medicine, London,
England; INSERM-CreS, Centre de Recherche d'Immunologie
et d'Hématologie, Strasbourg, France; and
Walther-Straub-Institut für Pharmakologie und Toxikologie,
Ludwig-Maximilians-Universität, München,
Germany.
 |
Abstract |
Hereditary hemochromatosis is a common iron-loading disorder found
in populations of European descent. It has been proposed that mutations
causing loss of function of HFE gene result in reduced iron
incorporation into immature duodenal crypt cells. These cells then
overexpress genes for iron absorption, leading to inappropriate
cellular iron balance, a persistent iron deficiency of the duodenal
mucosa, and increased iron absorption. The objective was to measure
duodenal iron content in Hfe knock-out mice to test whether
the mutation causes a persistent decrease in enterocyte iron
concentration. In both normal and Hfe knock-out mice,
duodenal nonheme iron content was found to correlate with liver iron
stores (P < .001, r = 0.643 and 0.551, respectively), and this effect did not depend on dietary iron levels.
However, duodenal iron content was reduced in Hfe knock-out
mice for any given content of liver iron stores
(P < .001).
(Blood. 2003;101:3316-3318)
© 2003 by The American Society of Hematology.
| |
Introduction |
Genetic hemochromatosis is a common hereditary
defect in human beings that can lead to massive tissue iron loading
with associated pathology1 due to inappropriate intestinal
iron absorption.2 Body iron levels in mammals are normally
tightly controlled by regulation of intestinal iron absorption. In
populations of Northern European descent, 80% of genetic
hemochromatosis is related to a Cys282Tyr mutation in the
HFE gene. In both humans and mice, null
mutations and Cys282Tyr mutation cause iron-loading
phenotypes,3-6 suggesting that the function of HFE gene
product is required for iron homeostasis.
Mice with targeted mutations in Hfe gene are an important tool for
understanding such genetic disorders, and several examples of mice with
disrupted Hfe expression have been studied.4,5 A similar
phenotype also occurs in 
2-microglobulin knock-out mice,
which fail to express cell surface Hfe protein.7 All these
mice develop increased liver iron, considered to be a characteristic of
hemochromatosis, although the degree of increase varies between mouse
genotypes.5 The best hypothesis to explain the
inappropriate iron absorption, seen when HFE function is
disrupted, suggests that plasma membrane HFE protein interacts with
transferrin receptor and 2-microglobulin to determine
iron levels in the duodenal crypt.8,9 HFE is expressed in
the crypt,10 but not on the villus, where dietary iron
uptake and iron absorption genes are expressed.11-13 A recent study has shown that
uptake of plasma radioiron into intestine (presumed to be mediated via
transferrin receptor, which is expressed mainly in crypt cells) is
indeed reduced in Hfe knock-out (KO) mice.14 If
the iron supply from the plasma to the immature crypt cells were a key
signal to gear intestinal iron absorption to body iron
stores,15,16 such inappropriately low iron uptake should
change the set point of this feedback loop and thereby explain its
dysregulation in hemochromatosis. To further test this hypothesis, we
set out to measure steady-state duodenal and hepatic iron in
sex- and age-matched Hfe KO and wild-type controls
fed various levels of dietary iron.
| |
Study design |
Hfe KO breeders (originally mixed 129/Ola
C57BL/6
background strain4,17; donated by Susan Gilfillan,
Department of Immunology, Washington University, St Louis, MO) were
mated with C57BL/6 and subsequently genotyped by polymerase chain
reaction (PCR).4 Wild-type and Hfe KO
homozygote breeders were established to produce age-matched male mice
for experimental study. Mice at 7 weeks of age were maintained on
either an iron-adequate (180 mg iron per kilogram) or iron-deficient (6 mg iron per kilogram) diet ad libitum for 5 more weeks as reported
previously.4 Mice were killed by pentobarbitone overdose,
and the duodenum was removed and rinsed in saline. All
experiments were carried out under the authority of the United Kingdom
Home Office. Tissue nonheme iron content was determined
as described previously.18 Correlation was
examined by linear regression.
| |
Results and discussion |
Deletion of the Hfe gene had no effect on body weight
but increased body iron stores as measured by liver nonheme iron (Table 1, P < .001). Iron
absorption remains sufficiently well regulated in Hfe KO mice with this
genetic background, so the high iron overload characteristic of human
hemochromatosis is not seen in them.17 This combined with
the large variation seen in these animals (M.-P. Roth, personal
communication, September 2002) is useful because it means that
correlations can be informative with some overlap between the KO and
wild-type genotype.
Duodenal iron was found to be correlated with liver iron in both
wild-type and Hfe KO mice (wild-type,
r = 0.587, P < .05, n = 12; Hfe
KO, r = 0.643, P < .01, n = 15) fed an
iron-adequate diet. However, the slope of the linear regression line
differed significantly for the 2 genotypes (P < .05). To
extend the range of iron stores studied and to investigate the impact
of dietary iron, mice were fed an iron-deficient diet in place of an
iron-adequate diet for 5 weeks, and the relationship between duodenal
and liver nonheme iron was determined. Both genotypes showed no
significant difference in the slope of the regression lines on an
iron-deficientcompared with an iron-adequatediet, demonstrating a
steady state in body iron distribution in both cases and that this
effect was not due to dietary iron level. It must therefore represent a
property of body iron metabolism, and data from both diets for each
genotype were combined (Figure 1). Mice
of both genotypes fed with the iron-deficient diet supplemented with 25 g/kg carbonyl iron had greatly elevated liver and duodenal nonheme iron
values (not shown) in line with previous findings.4
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| Figure 1.
Duodenal and liver iron content in mice fed an
iron-adequate or iron-deficient diet.
Data points are wild-type ( ,  ) and Hfe KO ( ,  )
mice fed iron-adequate (, ) or iron-deficient (, ) diets.
The lines are linear regression fits to data for both diets combined
with the following slopes: wild-type (bold line), 0.36 ± 0.09,
P < .001, r = 0.643, n = 24;
Hfe knock-out (faint line), 0.025 ± 0.007,
P < .001, r = 0.551, n = 33.
Comparison of regression slopes: P < .001.
|
|
It has been known for some years that duodenal iron changes when body
iron stores are manipulated (reviewed by Turnbull19). There has been debate about the cellular location of this iron, but
work in mice showed that changes in villus enterocyte iron reflected
changes in the whole mucosa.20 A relationship between iron
stores and duodenal iron is in line with the hypothesis for iron
absorption regulation proposed by Conrad and Crosby.15 It
is reported that hemochromatosis patients have duodenal iron content in
the normal range, despite their increased iron stores,21 and that their enterocytes are functionally iron
deficient.22
The present study found that wild-type mice fed an iron-adequate diet
exhibited a correlation between hepatic and duodenal iron. The 5-week
diet feeding regime ensured that a steady state in liver and duodenal
iron was reached.16 The measurements did not require
perturbation of iron metabolism prior to killing the mice. A
correlation was also found in Hfe KO mice; however, the slope of the line differed between the 2 genotypes. This has several implications; first, mice must have a mechanism that relates iron stores to duodenal iron, and Hfe KO mice retain such a
mechanism. Second, Hfe gene product acts to modulate this
relationship and is presumably necessary to ensure the correct "set
point"23 for duodenal iron. Third, villus enterocytes
must retain a "memory" of the iron levels experienced when they
developed, and in mice lacking Hfe protein duodenal iron is reduced
compared with wild-type mice with the same iron stores. Recently,
hepcidin24 has been implicated as a potential signaling
mechanism linking liver iron stores to duodenal iron absorption,
presumably operating in parallel with the crypt cells' own ability to
sense plasma iron.16 The present data suggest that such
mechanisms still function in Hfe KO mice, although inappropriately.
The question of how plasma iron changes are "remembered" by the
enterocytes as they develop remains a central issue in the regulation
of iron absorption.1,8,9,23 Interestingly, a recent study
by Oates et al25 provided evidence that small changes in
crypt cell iron are amplified as the enterocytes develop and move along
the villus, the function of divalent metal transporter 1 (DMT1) being necessary for this process by supplying iron from the
intestinal lumen. Schümann et al16 showed that
altered iron regulatory protein (IRP) activity could form part
of this memory. The work of Trinder et al14 points toward
reductions in iron uptake by the crypt being caused by loss of Hfe
function. These changes presumably lead to a setting of the balance
between uptake of luminal iron and its release into the plasma, which then persistently determines enterocyte iron.
| |
Footnotes |
Submitted October 15, 2002; accepted November 22, 2002.
Prepublished
online as Blood First Edition Paper, December 5, 2002; DOI
10.1182/ blood-2002-10-3112.
Supported by the Wellcome Trust, Sir Jules Thorn Charitable
Trust, and United Kingdom Medical Research Council (UK MRC).
S.B.'s laboratory is supported by Ministère de la Recherche and
INSERM of France.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Reprints: R. J. Simpson, Department of Life
Sciences, King's College London, Franklin Wilkins Building, Stamford
St, London SE1 9NN, England; e-mail:
robert.simpson{at}kcl.ac.uk.
| |
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Abstract
Introduction